We aim to study how neural activity at the level of single unit responses in the hippocampus mediates the known roles of synaptic plasticity and hippocampal neural circuitry in learning and memory. In particular, we are interested in the synaptic plasticity and circuit mechanisms responsible for the activation of hippocampal units in the rat in precise sequences that depict past and future behavioral trajectories. (1) Pioneering work has established that place field responses can reflect multiple aspects of memory and exhibit a complex dependence upon mechanisms of synaptic plasticity, and yet experience-dependent changes to place fields tend to be rather subtle and challenging to detect. In contrast, trajectory-specific place-cell sequences can be detected after very little experience. We will examine the dependence of this possible learning effect on mechanisms associated with synaptic plasticity, with particular attention to the interesting counter-hypothesis that the sequences may exist prior to experience. Preliminary data show that synaptic plasticity is required during experience in order to encode replay memory, whereas the generation of replay per se does not require synaptic plasticity if the underlying memories have already been formed. We will pursue these experiments to understand how multiple mechanisms of synaptic plasticity shape place-cell sequences. (2) A central role for CA3 in the generation of replay is a longstanding but untested prediction. Previous studies using genetic silencing of CA3 input found preserved ripple patterns in CA1, but these studies may have allowed compensatory effects due to the long time course of suppression. We will pursue an optogenetic approach for instantaneous suppression. Preliminary data show that ripples and ripple-associated spiking are in fact dependent upon CA3 input to the locally recorded CA1 region. We will also online decode replay sequences in real-time, to selectively disrupt sequences and also sequence subcomponents. We will dissect the contribution of CA3 to replay initiation, direction, propagation and termination. (3) During the theta exploratory state, CA1 units are driven by two major inputs: CA3 and entorhinal cortex (EC), which recent reports show may interact in complex ways, both within and across different theta cycles. These hypothesized interactions have not been tested directly, and so we will utilize our optogenetic approach to examine CA1 unit activity during this state. Preliminary data show that in contrast to ripples, CA1 spiking is only partially reduced when CA3 input is suppressed during theta, unmasking the EC contribution. We will examine place fields, phase precession, theta sequences, and the synchronization of CA1 low/high gamma with CA3/EC, either with or without CA3 input. Taken together, these specific aims represent a unique approach that utilizes the power of ultra-high density unit recording together with pharmacological and optogenetic manipulation, to deliver insights into the neural basis of learning and memory. Our results will have a major impact on understanding those diseases that impair hippocampal learning and memory such as Alzheimer's disease, epilepsy, stroke and normal aging.